title: Enzymatic characterization of a type II isocitrate dehydrogenase from pathogenic Leptospira interrogans serovar Lai strain 56601.
abstract: Leptospira interrogans, a Gram-negative pathogen, could cause infections in a wide variety of mammalian hosts, but due to their fastidious cultivation requirements and the lack of genetic systems, the pathogenic factor is still not clear. Isocitrate dehydrogenase (IDH) is a key enzyme in the tricarboxylation (TCA) cycle, which could have an important impact on the growth and pathogenesis of the bacteria. In the present study, we first report the cloning, heterologous expression, and detailed characterization of the IDH gene from L. interrogans serovar Lai strain 56601(LiIDH). The molecular weight of LiIDH was determined to be 87 kDa by filtration chromatography, suggesting LiIDH is a typical homodimer. The optimum activity of LiIDH was found at 60 °C, and its optimum pH was 7.0 (Mn(2+)) and 8.0 (Mg(2+)). Heat inactivation studies showed that heat treatment for 20 min at 50 °C caused a 50 % loss of enzyme activity. LiIDH was completely divalent cation dependent as other typical dimeric IDHs and Mg(2+) was its best activator. The recombinant LiIDH specificities (kcat/Km values for NADP(+) and NAD(+)) in the presence of Mg(2+) and Mn(2+) were 6,269-fold and 1,000-fold greater for NADP(+) than NAD(+), respectively. This current work is expected to shed light on the functions of metabolic enzymes in L. interrogans and provide useful information for LiIDH to be considered as a possible candidate for serological diagnostics and detection of L. interrogans infection.
authors.affiliation: Institute of Molecular Biology and Biotechnology and Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu, 241000, Anhui, China.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Zhao
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.forename: Guiyue
authors.initials: G
authors.lastname: Zhu
authors.forename: Baojuan
authors.initials: B
authors.lastname: Wang
authors.forename: Guoping
authors.initials: G
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D007921:Leptospira interrogans / Q000201:enzymology* / Q000502:physiology; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017434:Protein Structure, Tertiary; D017421:Sequence Analysis; D013696:Temperature
chemical_list: D008670:Metals; D007521:Isocitrate Dehydrogenase